Shipping fever vaccine



United States Patent SHIPPING FEVER VACCINE Charles Gale and Earl E.Ose, Indianapolis, Ind., as signors to Eli Lilly and Company,Indianapolis, Ind., a corporation of Indiana N0 Drawing.Continuation-impart of application Ser. No. 565,397, July 15, 1966. Thisapplication Apr. 13, 1967, Ser. No. 630,529

Int. Cl. A61k 23/00 US. Cl. 424-89 Claims ABSTRACT OF THE DISCLOSURE Avaccine of the inactivated virus type for immunizing cattle againstshipping fever prepared by mixing inactivated paraiinfluenza-3 virus andinactivated Pasteurella multocida and hemolytica bacteria cultures to aneffective McFarland density and absorbing these inactivated organisms onan aluminum ion adjuvant.

CROSS-REFERENCE This application is a continuation-in-part of ourcopending application, Ser. No. 565,397, filed July 15, 1966, nowabandoned.

BACKGROUND OF THE INVENTION This invention relates to a shipping fevervaccine of the inactivated or killed virus type. More particularly, thisinvention provides an improved killed virus-bacteria composition whichhas the capacity to provide substantially complete protection to cattleagainst bovine Myxovirus palainfluenZa-3 virus infection andPasteurellosis associated with shipping fever.

The occurrence of Myxovirus parainfluenza-3 virus and Pasteurellaorganisms in the respiratory tract of cattle has been well documented.That these agents are important etiological agents in the shipping fevercomplex is now well established. The etiological factors in shippingfever are apparently a complex of several elements. The hypothesis of avirus, perhaps latent, being activated by the stress of shipment to makea favorable environment for secondary bacteria finds some support inexperimental studies conducted by those in the veterinary medicalscience. It is well established that Myxovirus parainfluenza-3 virusesare widely distributed and result in a spectrum of clinical disordersvarying from mild subclinical disease to severe disease that can resultnot only in clinically apparent respiratory signs but death of theanimal. A considerable amount of data indicated that Pasteurella speciesplays a role in shipping fever have appeared in the literature.

'The vaccines prepared before this invention, in attemps to find aneffective immunizing agent for healthy cattle against the shipping feverdisease or complex, have not been satisfactory in that they haveprovided inadequate protection. In the Cornell Veterinarian, vol. 54, 1(January 1964), in an article entitled Experimental Immunization ofCattle Against Shipping Fever, it was concluded that aforrnol-inactivated vaccine composition in which the virus and bacterialportions were concentrated separately and mixed with an oil adjuvantgave inadequate protection. Thus, earlier vaccines have failed toprovide the desired immunization in cattle. The cattle raising industryis stillseeking a satisfactory immunizing composition for use inpreventing the effects of the shipping fever complex in cattle.

SUMMARY OF THE INVENTION It is an object of this invention to provide aneffective shipping fever vaccine.

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Another object is to provide a method for preparing a satisfactoryinactivated virus-bacteria composition which methods in a tissue cultureof fetal or adult bovine,

simian, rabbit or similar monolayer submerged kidney cells, eitherprimary cells or a cell line, until the virusinoculated cells exhibit acytopathogenic effect characteristic of this strain of virus.

In a separate operation, Pasteurella multocida and Pasteurellahemolytica are separately grown in agar or liquid nutrient media. Ifdesired, the virulence of the bacteria may be increased by first growingthe bacteria in chicken embryo allantoicamnionic sac tissue or intissues of mice, rabbits, or guinea pigs. Each of these bacteria is thenseparately harvested from its growth medium, by commingling portions ofvirus harvest fluid With the respective bacteria-containing media toobtain separate live virus-P. m ult0cida and virus-P. hemolyticabacteria mixtures. The separate virus-bacteria mixtures are adjusted toabout equal density within the range of from about 5 to 10 timesMcFarland tube number one, as measured by the standard McFarland method,and then portions of the separate virus-bacteria mixtures arecommingled. The pH of the combined virus-P. multo'cida and P. hemolyticabacteria mixture is adjusted to about 661-02, and then the mixture iscommingled with an aluminum ion-containing adjuvant by suitable mixingprocedures.

The adjuvant-containing virus-bacteria composition is then inactivatedby the addition of formaldehyde. After the formaldehyde-treatedvirus-bacterial mixture has been inactivated, any excess formaldehyde isneutralized with a suitable sulfite salt solution. Samples of theinactivated virus-bacterial composition are set aside forbacteriological and virus antigenicity tests and then the bulk of theproduct is preserved by treating it with a suitable parenterallyacceptable antiseptic in bacteriostatic and fungistatic amounts. Theresulting inactivated virus-bacteria-adjuvant composition may be used assuch or can be concentrated by decanting supernatant liquid to yield aproduct of increased potency.

Alternatively, the Myxovirus parainfluenzai-B culture, the P.hemolytica: culture, and the P. multociaa culture may be separatelyinactivated with formaldehyde-containing media, and'then mixed, eitherbefore or after adding the aluminum ion adjuvant to the virus-containingculture. We prefer to add the aluminum ion adjuvant to the inactivatedvirus culture before the inactivated P. hemolytica and P. multocidacultures are added. This allows separate tests to be made with virus-P.hemolytica! and virus-P. multocida mixtures to determine the proportionsof virus culture and P. hemolytica and P. multo'cida cultures necessaryto obtain optimum virus-bacterium mixtures. After the aluminum ionadjuvant has been mixed with the virus culture, the calculated amountsof inactivated P. hemolytica and P. multocida cultures can be added toobtain the vaccine composition of the de-- characteristic cytopathogeniceffect in bovine kidney tissue cultured cells in 2 to days. The virusmust be neutralized by specific parainfluenza-3 antiserum. The virusmust cause hemagglutination (HA) of guinea pig red blood cells. Thevirus must also cause hcmadsorption of guinea pig red blood cells ininfected cultures. The viruscontaining seed cultures used in practicingthis invention should have a virulence HA titer of 1:40 or higher onguinea pig red blood cells and have an infectivity titer in bovinekidney tissue cultures of per milliliter or higher. The virus culturesare routinely checked for bacteriological sterility and freedom fromPleuropneumoniadike organisms.

The virus seed and production cultures are propagated in primary or cellline or diploid cell strains from embryonic or adult bovine or simiankidney tissues, which are prepared by trypsinidation of the tissues anddispersion of the cells in Hanks or Earles balanced salt 'solutionenriched with a small amount of lactalbumin hydrolysate. The productionculture medium may contain bovine serum if desired, but it is notessential. Small amounts of penicillin and streptomycin can be used inthe mixture as preservatives. The vessels used can be of any appropriatesize. We have grown the virus in vessel sizes ranging from 2-ounceprescription bottles to 5-liter Pyrex Povitsky bottles. The virus seedculture is lyophilized with sterile distilled water and suitablestabilizer.

The production culture medium is inoculated with the lyophilized seedculture aseptically.

The seed volume is generally about 4 percent to 6 percent of theproduction medium volume.

The virus is grown in this medium until a titer of at least 10 virusinfected cells per milliliter is obtained. We

prefer to grow the virus until the titer is about 10 virus infectedcells per milliliter. By way of example, a suitable virus titer may beobtained by incubating a virus-inoculated primary bovine kidney cellmedium at about 38 C. for 2 to 7 days.

Only those virus-containing vessels which are visibly free of bacteriaor other contamination are considered for the virus harvest.Satisfactory virus-laden tissue culture fluids are harvested byaseptically transferring the culture fluids, including the cells,directly to the bacteria mixing step or to suitable pooling containersfor use in washing each bacteria growth medium. This usually takes placefrom about 2 to 7 days after inoculation of the growth media. The livevirus-containing fluid can be used immediately or, optionally, may befrozen and stored at refrigerator or freezer temperature to increase thevirus harvest obtained, and to maintain the virus for later use. Avirus-containing culture fluid must have an HA titer of 1:40 or higherwith guinea pig red blood cells and have virus infectivity titer inbovine kidney tissue cultures of at least 10 virus particles permilliliter of medium to be satisfactory for use as a bacteria wash fluidin this invention.

' As indicated above, the virus-containing tissue culture may first beinactivated with formaldehyde before mixing it with the bacterialcultures.

Seed cultures of Pasteurella organisms may be grown in commerciallyavailable nutrient media by conventional methods. In producing largerquantities of each bacterium, a lyophilized vial of each bacterialstrain is used to inoculate large volumes of agar or tryptose broth tobe used as production inoculum after 7 to 24 hours incubation. Thequantity of seed can vary depending upon the vessel used. Usually enoughof the bacterial broth seed culture is used to coverthe agar surface.When liquid culture media are used, the medium is inoculated with fromabout 1 to 5 percent by volume of seed in the production broth culture.The bacterial cultures are incubated at about 35-38 C. for a timesufficient to allow the bacterial titer to grow to at least 10",preferably 10 viable bacterial cells per milliliter.

Separate live P. hemolytica and P. multocida cultures may be mixed withlive parainfluenza-3 virus tissue culture fluid, and then the mixtureinactivated with formaldehyde. Alternatively, the P. hemolytica and P.multocida cultures may be separately inactivated by the addition theretoof suitable formaldehyde solutions, stored until ready for use, and thenmixed with the virus tissue culture. In mixing the virus tissue culturewith the bacterium cultures, care is taken in the proportions of thevirus culture fluid and bacterium culture fluids used to insure that thefinal virus-bacteria fluid density is equivalent to the above-indicatedMcFarland range. Formaldehyde inactivated virus-bacterium culturemixture may also be mixed to provide a composition of the requiredMcFarland den- After mixing the virus tissue culture fluid with eachbacterium culture, fluid, each virus-bacteria mixture can be adjusted tosubstantially the same density by addition of virus tissue culture fluidor bacteria culture as needed. Using the McFarland density measurementscale as a reference, we prefer to adjust the density of eachvirus-bacteria mixture to about 7 to 8 times McFarland tube number one,although any density between about 5 and 10 times McFarland number onemay be used. If the Pasteurella cultures are grown in a liquid medium,enough of the virus tissue culture fluid is used to dilute theindividual Pasteurella pools to the desired final density. ThePasteurella culture should not represent more than about 25 percent ofthe volume of the virus-bacteria mixture.

After adjusting the separate M. parainfluenza-3 virus-P. multocida andM. parainfluenza-3 virus-P. hemolytica mixtures to the desired density,substantially equal volumes of the separate pools are commingled, andthe pH of the resulting mixture is adjusted to 6.4 to 6.8 by suitableaddition of an acid, such as 0.1 N hydrochloric acid or a base such assodium hydroxide as necessary. To the pH-adjusted mixture thus obtained,the aluminum ion-containing adjuvant is added. Alternatively, the virusculture, the P. hemolytica culture, and the P. multocida culture may beseparately inactivated with formaldehyde solution. After determining theproportions of virus culture and P. hem0- lytica culture, and virusculture and P. multocida culture which give a composition of the desireddensity as determined, e.g., by the above-described test methods, theinactivated virus culture may be mixed with the aluminum ion adjuvantmixture before the determined amounts of P. hemolytica and P. multocidacultures are mixed with the virus culture.

The aluminum ion-containing adjuvant used must be one which contains thetrivalent aluminum ion in a pharmaceutically acceptable form. It may bein the form of an aqueous solution of a suitable aluminum salt, anadsorptive solid, or an aqueous aluminum ion-containing gel. Examples ofsuitable aluminum ion forms which may be used include aqueous aluminumhydroxide or aluminum oxide gels or aqueous dispersions, and aluminumsalts and mixed salts of aluminum and various acids. Preferred forms ofthe aluminum ion-containing material are an aqueous aluminum hydroxidegel, an aqueous active alumina suspension, and a hydrated aluminumphosphate, in freshly precipitated form for greatest activity. Thesematerials may be prepared by metathesis of suitable precursor salts inaqueous solution orsuspension, and then filtering and washingthe-resulting precipitate, suitable precautions being taken to insuresterility before use. Aluminum phosphate can be prepared, for example,by commingling aqueous solutions of aluminum chloride and disodiumhydrogen orthophosphate, or aluminum potassium sulfate and trisodiumorthophosphate. The resulting precipitate is filtered off and washedwith water to remove soluble materials, and then can be added in the wetstate to the inactivated virus culture or to the virusbacteria mixtureas described above. The aluminum ioncontaining material is commingledwith'the inactivated virus-culture or virus-bacteria mixture in anaqueous solu tion or gel or absorptive solid dispersion having analuminum compound concentration of from about 0.5 percent toabout 5percent by weight, preferably from about 1 to 2 percent by weight, andis added in an amount to be chemically equivalent to from about to 50milliliters, preferably 10 to 30 ml. of an aqueous 1.3 percent by weightof aluminum oxide (Al' O per 100 ml. of virus bacteria culture mixture.The resulting mixture should be agitated to insure uniform composition.A -minute stirring is usually sufficient for this purpose.

For the inactivation, or virus-bacteria killing step, formaldehyde isadded. The formaldehyde solution is added in an amount to provide aconcentration sufiicient to inactivate the vims and-bacteria Withoutmaterially impairing the antigenicity of the resulting vaccine. Usuallyabout 0.075 to about 0.125 percent by weight of formaldehyde issufficient for this purpose. It is understood, however, thatformaldehyde can be employed in any form which provides the activealdehyde for contact with the virus and bacteria, e.g., trioxymethylene,solid polymeric formaldehyde, or formaldehyde condensation products andthe like which release formaldehyde in aqueous media. It will be equallyapparent that the formaldehyde composition should not include substanceswhich would be detrimental to the vaccine composition if they must beallowed to remain in the composition.

The inactivation of the virus and bacteria appears to be essentiallycomplete in about six hours. As a precautionary measure however, theformaldehyde treated cultures are preferably held for at least about 24hours, but not more than about four days at room temperature. Otherinactivating agents which may be used to replace part or all of theformaldehyde are beta-propiolactone, hydroxylamine, phenol, andchloroform and the like. Formaldehyde is preferred however.

After the holding period, the composition can 'be concentrated, ifdesired, by removing a portion of the supernatant liquid. Excessformaldehyde in the resulting vaccine composition is then neutralizedwith a suitable formaldehyde absorber. A convenient neutralization agentfor this purpose is a 10 to 35 percent sterile aqueous sodium bisulfitesolution. If desired, the excess formaldehyde may be neutralized withthe sodium bisulfite prior to mixing the virus composition with thealuminum ion adjuvant. Samples of the resulting product are withdrawnfor Standard Requirement P-l5 bacteriological sterility testing andvirus inactivation safety testing, and then the mixture is treated witha suitable parenterally acceptable antiseptic as a bacteriostatic andfungistatic preservative. Examples of antiseptics which may be usedinclude chloroform, phenol, and thimerosal. We prefer to add thimerosalto a concentration of about 0.4 ml. to about 0.6 ml. of a 10 percentsolution per liter of bulk product, but other antiseptics may be used.The completed bulk vaccine can be packaged immediately or stored atrefrigerator temperature pending final container filling.

Viral safety testing is accomplished by the inoculation of six tubes ofbovine tissue culture monolayers with 0.2 ml. of a 1:30 dilution of thebulk vaccine product, taken prior to the addition of the antisepticpreservative. These six tubes with adequate controls, are observed for 7to 9 days. If no cytopathogenic effect is found and the controls remainessentially normal, the virus is considered to be inactivated.

The potency of the composition can be measured by several methods. Onesuch test method, which we term the hemagglutination inhibition (HI)activity method, involves the selection of six calves, 3 to6 months ofage, which have blood serum HI activity titers lower than 1:20, asmeasured by standard procedureswith guinea pig red blood cells, and theinoculation of three of these calves United States Department ofAgriculture Regulation (Agriculture Research Service, Animal Inspectionand Quarantine Division), P45 Standard Sterility Test Procedures forVeterinary Biological Products.

with two intramuscular injections of IO-cc. doses of thevaccine-bacterin product with an interval of 2 to 3 weeks betweeninjections. All six calves are housed together during the test period.Samples of blood are taken from the six calves 2 to 3 weeks followingthe second injection. Comparative HI activity titer testing of preandpostvaccination blood serum samples must demonstrate a serum HI titerconversion to 1:80 or higher in the vaccinated calves with nosignificant increase in the serum titer of the unvaccinated calves. Thepotency of the composition may also be determined by challenge tovaccinated and unvaccinated calves with a combination of M.parainfluenrza-3 virus and cultures of P. multocida and P. hemolyticagiven by the aerosol route or intratracheally or by other suitableroute. Signs of shipping fever disease such as increased temperature,cough and nasal discharge must not occur in the vaccinated calves andmust occur in the control unvaccinated calves. Potency can also bedetermined by a suitable serum neutralization (SN) test or bycorrelating HI titer conversions in guinea pigs. Serological conversionin guinea pigs is indicated by demonstration of a 10-fold increase ingeometric mean titer (GMT) in vaccinated guinea pigs with no significantincrease in the GMT in the unvaccinated control guinea pigs.

The Pasteurella antigenic properties of the vaccinebacterin compositionof this invention can be established by the following test method:

Swiss mice weighing 15 to 20 g. are each given two 0.2 ml. subcutaneousinjections of finished bulk product. The interval between injections is1014 days. Seven to 14 days following the second injection, equalnumbers of vaccinated and unvaccinated mice are challenged with tenfolddilutions of virulent P. multocida broth culture. Both vaccinated andcontrol mice are divided into dilution groups of 5 or more mice. Atleast 2 dilution groups are used for vaccinated mice and for controlmice. The post-challenge observation period is seven days. Fifty percentmortality end points (LD' are calculated for both vaccinated and controlmice according to the method of Reed and Muench. The test is consideredsatisfactory if the LD in vaccinated mice is at least two logs lowerthan that demonstrated in the unvaccinated mice.

The dosage recommended for cattle is at least one dose of from 2 to15-cc. injected intramuscularly or subcutaneously. We prefer toadminister two doses of about 10 cc. with an interval of about 1 to 4weeks between injections.

The vaccine composition of this invention may be incorporated intomultiple antigen combinations to permit simultaneous immunization of thecattle against two or more diseases. Such combinations can be made, forexample, with. antigens for Brucella sp., Clostridium sp., bovine virusdiarrhea virus (BVDV), infectious bovine rhinotracheitis, (IBR) and thelike. i

More in detail, the invention is illustrated by the following examples.

EXAMPLE 1 This example illustrates the preparation of the vaccinebacterin composition of this-invention.

Virus preparation Production cultures of the M. parainfluenza-3 weregrown in 5-liter Pyrex Povitsky bottles. The seed volume used wasapproximately 5 percent of the production flask medium volume. Theculture flasks containing the seed were incubated at 35-37 C. for 3 to 5days, or until approximately 65 percent of the inoculated cells wereexhibiting cytopathic effects characteristic of the SP4 strain of virus.Satisfactory virus-laden tissue culture fluids including cells wereaseptically transferred to pooling containers. The tissue culture virusharvest, collected from 2 to 7 days after inoculation, was then used, asindicated below, to collect the two different Pasteurella cultures fromagar.

Pasturella bacteria portion Seed cultures of each of Pasteurellamultocida P1062 and Pasteurella hemolyrica P1148 strains were separatelygrown for 8 to12 hours at 37 C. in tryptose broth. These bacteria seedcultures were inoculated in 0.1-ml. volumes into allantoic-amnionic sacsof 8 to 10-day old embryonating chicken eggs. After approximately 24hours incubation (at which time one-half of the embryos were dead), theeggs were chilled and the allantoic-amnionic fluid harvested and pooled.Each pooled strain of bacteriacontaining fluid was lyophilizedseparately. These cultures were used as the seed inocula.

A vial of each of the above bacteria strains was prepared in tryptosebroth (Difco). After 10 to 12 hours incubation at 37 C., these cultureswere used to inoculate tryptose broth to use as an inoculum for agar.After 12 to 15 hours incubation, separate agar substrates wereinoculated using sufiicient inoculated tryptose broth culture to coverthe entire agar surface. The agar used was a Difco starch agar with anadditional 1.5 percent agar added. These production agar cultures wereincubated at 37 C. for 15 to 18 hours after inoculation.

The bacteria-containing production culture flasks were harvested after12 to 48 hours by washing the bacteriacontaining agar cultures withsufiicient M. parainfluenza-3 harvest tissue culture fluid to remove thebacteria from the agar. The washings were conducted in such a mannerthat a mixture of virus and P. hemolytica and a mixture of virus and P.multocida were separately prepared. The density of each mixture wasadjusted to 7.5 times Mc- Farland tube number one by the addition of M.parainfluenza-3 harvest tissue culture fluid and then equal volumes ofthe mixtures were commingled. The combination product was adjusted to pH6.6102 by adding 0.1 N hydrochloric acid and then mixed with aluminumhydroxide gel (calculated as containing 1.3 percent A1 0 at the rate of20 ml. of the aluminum hydroxide gel per 100 ml. of the combinationproduct culture mixture. The gel-culture mixture was shaken for 15minutes to insure complete distribution. Formalin (38 percent aqueousformaldehyde solution) was added as a 10 percent formaldehyde in watersolution to give a 0.1 percent formalin concentration and the mixturewas shaken for another 15 minutes. The formaldehyde-treated mixture wasstored and allowed to react for 24 hours at room temperature, and thenthe composition was concentrated by decanting approximately one-half ofthe supernatant fluid. The excess formaldehyde in the remaining combinedvirusbacteria-alumina gel mixture was neutralized with 35 percentsterile aqueous sodium bisulfite and then samples of the product wereremoved for Standard Requirement P-15 bacteriological sterility testingand to insure inactivation of the virus and bacteria contained therein.Merthiolate brand of thimerosal (10 percent solution) was added to thebulk product at the rate of 0.5 ml. per liter. The completed bulkproduct, thus obtained, was stored at refrigerator temperature pendingfinal container filling.

The viral safety testing was accomplished by the inoculation of sixtubes of bovine tissue culture monolayers with 0.2 ml. of a 1:50dilution of the bulk product (prior to the addition of Merthiolate).These six tubes, with adequate controls, were observed for 7 to 9 days.No cytopathic effect (CPE) was found in the inoculated vials, and thecontrols remained esesentially normal. The virus was thus considered tobe inactivated.

EXAMPLE 2 Four lots of vaccine compositions prepared as described inExample 1, were tested in bovine calves and guinea pigs in both aconcentrated (one-half of supernatant liquid decanted off before NaHSOneutralization) and unconcentrated form, using the hemagglutinationinhibition (HI) titer test method.

In this test bovine calves were inoculated by intramuscular injectionswith two 10-cc. doses of the vaccine product with three weeks betweeninjections. Some calves were inoculated with the unconcentratedcomposition; others were inoculated with the concentrated vaccine.Guinea pigs were given equivalent doses, of 1 milliliter, with the sameinterval between injections. Suitable uninoculated control calves andguinea pigs were maintained. Hemagglutination inhibition titer resultswere collected from all the calves and guinea pigs. The titer values forthe four lots of vaccine tested are summarized in the following table.

TABLE I Titer Guinea Guinea Pigs 2 Figs 2 Lot No Vaccine Calves 1(Nebraska) (Indiana) 1 None (Control) 3 7 22 4 5 Not concentrated 160 49132 Concentrated 508 113 236 2 None (Control) 0 9 Not concentrated 80732 132 Concentrated 508 60 539 3 N0ne(Contr0l) 25 10 Not concentrated807 103 321 Concentrated 320 108 906 4 None (Control) 160 11 Notconcentrated 807 297 202 Concentrated 255 260 260 1 Mean of 3 calves pervalue.

2 Mean of 8-10 guinea pigs per value.

3 Each number represents the second number in the HI titer ratio 1:Nwhere N is the number in the table.

4 Only one pen of controls was run for the Indiana tests but all fourlots were tested at the same time.

These data and other data illustrate the potency of the vaccinecomposition of this invention and that the guinea pig as well as thecalf can be used for measuring response to the vaccine. This finding wasconfirmed by statistical analysis.

' EXAMPLE 3 This example illustrates the preparation of a shipping fevervaccine composition. This procedure can be used to prepare the vaccineon a commercial scale.

Parainfluenza-3 virus medium and growth procedure Trypsinized fetalbovine kidney tissue culture (BKTC) cells, extracted from cortexportions of the kidneys were suspended in an appropriate BKTC growthmedium, incubated at 35 C. to 37 C. until the BKTC cells were confluent.The supernatant growth medium was discarded and replaced with similargrowth medium containing 1 ml. of live parainfluenza-3 (SF-4 strain)seed virus for each 99 m1. of growth medium added. The virus inoculatedBKTC tissue cultures, thus obtained, were incubated at 35 C. to 37 C.until the observable cytopathogenic effect (CPE) reaches 65 percent topercent, usually 48 to 96 hours after virus inoculation. Theviruscontaining cell media from several vessels were pooled. Afterremoval of samples for HA titer determination, virus titration, anddensity tests, the bulk of the live parainfluenza-3 virus containingBKTC medium was inactivated by treatment with enough dilutedformaldehyde solution (formalin, of commerce) to provide a concentrationof. about 1 part formaldehyde to 1000 parts of the virus suspension.(The formaldehyde solution was fur- 9 ther diluted to percentconcentration with a BKTC maintenance medium before adding to the viruscontaining medium.) The formaldehyde treated parainfluenza-3 virusmedium was stirred vigorously for about minutes.

The growth medium used for growing the cells was a mixture of 24 g. ofphenolsulfonphthalein sodium salt, 8.160 kg. of a sodium chloride, 480g. of potassium chloride, 246 g. of magnesium sulfate, 172.8 g. ofmonobasic sodium phosphate, 1.2 kg. of dextrose, 318 g. of calciumchloride, 6 kg. of lactalbumin hydrolysate, 672 g. of sodiumbicarbonate, 8.3 g. of penicillin G crystals, 120 g. of streptomycinsulfate, and sufiicient water to make 1200 liters. The mixture wasstirred for 10 minutes, sterilized at 250 F. for 4 hours and cooled toroom temperature and 24 liters of bovne serum added before use.

The maintenance medium used contained the same ingredients as the growthmedium, except that the maintenance medium contained nophenolsulfonylphthalein, contained 2.664 kg. of sodium bicarbonateinstead of the 672 g. of sodium bicarbonate in the growth medium, andcontained no bovine serum.

Pasteurella hemolytica P. hamolytica seed culture was first grown ontryptose and thiamine agar plates, incubated at 35 C. for 18 to 36hours, and then used to inoculate the P. hemolytica to tryptose andthiamine broth cultures. The P. hemolytica was incubated in the brothcultures for 6 to 18 hours at 35 C. After checks to insure againstcontamination, the contents of several P. hemolytica containing brothcultures were pooled in sterile containers and used as seed for largercultures of the organism. The P. hemolytica may be grown in a rollerdrum apparatus if desired, or as in this example in large bottles whichare shaken during incubation. The growth medium was inoculated with P.hemolytica seed culture equivalent to about 3 percent of the productiongrowth medium volume. The production cultures were incubated for 18 to24 hours at 35 C. on a shaking apparatus. The production cultures weretransferred to sterile containers and the density of the P. hem'olyticaculture was determined using a Cenco photolometer with a green filter,and the meter was set to 100 percent light transmission using a sterilemedium for the P. hemolytica. This Cenco photolometer was previouslycorrelated to reproduce the densities of from about 7 to 8 on theMcFarland scale.

The P. hemolytica thus obtained was inactivated by adding formaldehydesolution thereto. After incubation of the formaldehyde inactivated P.hemolytica culture for 48 to 72 hours at 35 C., the inactivatedbacterium was stored in a chill room (5 C.) until ready for use inpreparing the vaccine composition of this invention.

The growth medium used for growing the P. hemolytica consisted of 3.6kg. of NZ Case brand of a powdered protein and vitamin composition madeby tryptic digestion of casein, 1.8 kg. of yeast extract, 1.2 kg. oftryptone, 120 g. of magnesium sulfate, 324 g. of monobasic potassiumphosphate, 912 g. of dibanic sodium phosphate, 960 g. of dextrose in 90liters of water and warmed to 45 C., to which was added yeastautolysate. The pH of the medium was adjusted to 7.2 to 7.3 using 1normal sodium hydroxide solution, and then distilled water was added tomake 120 liters.

Pasteurella multocida P. multociaa: was propagated as described abovefor P. hemolytica, except that the growth medium contained 480 g. ofsucrose instead of the dextrose. After incubation and growth of the P.multocida and determination of density, using the Cenco photolometer,formaldehyde solution was added to the P. multocida culture toinactivate the bacterium.

Separate tests were first made with small sample mixtures of theinactivated parainfluenza-3 virus culture and 10 inactivated P.hemolytica culture to determine the amounts of each needed to give amixture which had the desired optical density on the McFarland scale.

Before mixing theinactivated virus with the inactivated bacteriumcultures, the virus culture was mixed with 360 ml. of a sterilized 1.3percent aluminum oxide suspension for each liter of virus culture andagitated for 15 minutes. Then the required amounts of the inactivated P.hemolytica and P. multocia'a cultures are added to the aluminum oxidetreated inactivated virus culture and mixed for 15 minutes. Theresulting composition was stored at 30 C. for 24 hours, and then thesupernatant liquid was decanted to remove a volume equivalent to thetotal volume of the added bacterial cultures plus 10-30 percent of thevolume of the total volume of the virus culture and aluminum oxidesuspension mixture.

The formaldehyde in the resulting concentrated, inactivatedparainfluenza-3 virus-P. hemolytica-P. multocida composition containingaluminum ion was neutralized with 35 percent sodium bisulfite solutionto a Schiifs Reagent end point. While agitating the mixture Merthiolatebrand of thimerosal solution for biologicals was added aseptically at arate of 0.5 ml. per liter of vaccine. Samples were removed fordetermination of bulk sterility, P. multocida potency (mouse), viruspotency (guinea pig), and virus potency (calf). The samples were storedat 5 C. until submission for test. The bulk of the vaccine was stored at5 C. until approval for packaging.

The aluminum oxide suspension used above was pre pared as follows:

Commercially available aluminum oxide gel was diluted with distilled,pyrogen-free water and the resulting suspension was passed through anEppenbach mill to reduce the gel particle sizes. The aqueous gelsuspension thus obtained was sterilized and then cooled to roomtemperature. The final aluminum oxide gel concentration was calculatedas containing the equivalent of approximately 1.3 percent aluminum oxide(Al O by weight.

We claim:

1. A method for preparing a shipping fever vaccine composition whichcomprises:

(a) growing Myxovirus parainfluenza-3 in bovine or simian kidney tissueculture;

(b) separately growing Pasteurella multocida and Pasteurella hemolyticain nutrient media;

(0) separately harvesting each bacterium from step (b) by comminglingvirus-laden tissue culture fluid from step (a) with each bacteriumculture to obtain separate virus-Pasteurella multocida bacterium andvirus-Pasteurella hemolytica bacterium mixtures;

(d) adjusting the density of the separate virusbacterium mixtures fromstep (c) to about equal density equivalent to from about 5 to 10 timesthe optical density of McFarland tube number one on the McFarland testmethod scale by adding virus tissue culture fluid or bacterium cultureas necessary;

(e) mixing the separate virus-bacterium mixtures from (f) adjusting thepH of the resulting combined virusbacteria mixture from step (e) toabout 6.4 to 6.8;

g) commingling the pH adjusted combined virusbacteria mixture from step(f) with an aluminum ion adjuvant selected from the group consisting ofaluminum hydroxide gel, aluminum oxide dispersion, and aluminumphosphate in an aluminum ion concentration equivalent to from about 10to about 30 ml. of an aqueous 1.3 percent A1 0 per ml. of the combinedvirus-bacteria mixture;

(h) inactivating the virus content of the combined virus-bacteriamixture from step (g) by commingling formaldehyde therewith;

(i) neutralizing any excess formaldehyde in the formaldehyde treatedvirus-bacteria mixture from step (h) 11 with an aqueous alkali metalbisulfite solution; and

(j) adding a parenterally acceptable antiseptic thereto inbacteriostatic and fungistatic amounts.

2. An improved shipping fever vaccine prepared by the method of claim 1.

3. A method for preparing an improved shipping fever vaccine compositionas described in claim 1, which includes a step of freezing thevirus-laden tissue culture fluid from step (a) before commingling itwith the bacterial cultures in step (c).

4. An improved shipping fever vaccine composition, prepared by themethod of claim 3.

5. A method as described in claim 1 wherein in step (a) the virus isgrown in a bovine kidney tissue cell culture until from about 50 toabout 70 percent of the cells exhibit the characteristic cytopathogeniceffect;

in step (c) the bacterial cultures do not exceed about 25 percent byvolume of the separate virus-bacterium mixtures;

in step (d) the density of the separate virus-bacterium mixtures isadjusted to between about 7 and 8 times the McFarland number one tubedensity;

in step (g) the aluminum ion adjuvant used in an aqueous aluminumhydroxide gel dispersion containing from about 1 to about 2 percent byweight of the aluminum hydroxide gel;

in step (b) the formaldehyde commingled with the combined virus-bacteriamixture is used in an amount to provide a formaldehyde concentration offrom about 0.075 to about 0.125 percent by weight; and in step (i)thimerosal is commingled with the inactivated virus-bacteria compositionin a concentration of from about 0.4 ml. to about 0.6 ml. of a 10percent thimerosal solution per liter of bulk product.

6. An improved shipping fever vaccine composition prepared as describedin claim 4.

7. A method of immunizing cattle against bovine Myxovirusparainfluenza-3 virus infection and Pasteurellosis associated withshipping fever which comprises injecting cattle with at least one doseof from about 2 to cc. of the improved shipping vaccine composition ofclaim 2.

8. A method of immunizing cattle against bovine Myxovirusparainfluenza-3 virus infection and Pasteurellosis associated withshipping fever which comprises injecting cattle With two doses,approximating at least about 2 to 15 cc. of the improved shipping fevervaccine com- 12 position of claim 5 at an interval of at least about 1to 4 weeks between injections.

9. A method which comprises (a) growing Myxovirus parainfluenza-S inbovine or simian kidney tissue cultures;

(b) treating the virus-containing bovine or simian kidney tissuecultures, from step (a) with formaldehyde to inactivate the virus;

(c) separately growing Pasteurella multocida and Pasteurella hemolyticain nutrient media; I

(d) treating the Pasteurella multocida and Pasteurella hemolytica fromstep (c) with formaldehyde to inactivate these respective bacteria;

(e) commingling inactivated virus cuture from step (b) with an aluminumion adjuvant selected from the group consisting of aluminum hydroxidegel, aluminum oxide dispersion, and aluminum phosphate in an amountchemically equivalent to an aluminum ion concentration of from about 10to about ml. of an aqueous 1.3 percent A1 0 mixture per ml. of theinactivated virus culture;

(f) mixing with the aluminum ion adjuvant containing inactivated virusculture with inactivated Pasteurella hemolytica and Pasteurellamultocida cultures from step (d) in amounts sufficient to provide aresulting inactivated virus-bacteria composition having a density offrom 5 to 10 times the optical density of McFarland tube number one onthe Mc- Farland test method scale.

(g) neutralizing with aqueous alkali metal bisulfite solution excessformaldehyde in the resulting composition from step (f), and

(h) adding a parenterally acceptable antiseptic thereto inbacteriostatic and fungistatic amounts.

10. An improved shipping fever vaccine prepared by the method of claim9.

References Cited Gale et al., J.A.V.M.A. 142(8): 884-887, Apr. 15, 1963.

King et al., J.A.V.M.A. 142(8): 881-883, Apr. 15, 1963.

Mohanty et al., Am. J. Vet Res. 25: 1653-1657 (1964).

Hamdy et al., Am. J. Vet. Res. 25: 1648-1652 (1964) Hamdy et al.,Cornell Vet. 54: 41-49 (1964).

Matsuoka et al., Proc. 69th Ann. Meet. U.S. Livestock Sanit.Assn.--l965, pp. 104-108 (1966).

S. K. ROSE, Primary Examiner

